Mosquito-borne diseases continue to have a significant impact on human health worldwide. However, with the presence of the Dengue fever and Chikungunya viruses and the biological vector Aedes aegypti in the United States, local impacts on human health are starting to be observed. Both the Dengue fever and Chikungunya viruses exclusively utilize humans as reservoirs and amplification hosts. This is important because the female Ae. aegypti mosquito requires a blood meal from a human host in order to obtain the proper nutrients to fuel the gonotrophic cycle. Unfortunately, no vaccines or treatments are available to combat the Dengue and Chikungunya viruses, and so the only strategy to prevent the spread of these viruses is through mosquito population (vector) control. Current vector control strategies using insecticides have proven effective in reducing the mosquito population and slowing down pathogen transmission. However, there has been an increase in Ae. aegypti mosquito resistance to available insecticides and with increasing world population, urbanization, and the lack of effective mosquito control strategies, the mosquito population will continue to grow. As a result, we will likely experience higher incidences of Dengue and Chikungunya infections. Therefore, our strategy is to target the proteolytic enzymes involved in blood meal protein digestion. The female Ae. aegypti mosquito relies on nutrients released by the digestion of blood meal proteins for egg maturation and production. However, before we can validate these midgut proteases as a potential vector control strategy, we must first fully understand how individual midgut proteases digest blood meal proteins. Our research will advance the knowledge of Ae. aegypti midgut proteases by biochemically studying the four most abundant midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). Aim 1.1 will focus on optimizing the soluble expression and purification of recombinant mosquito proteases, followed by biochemical and kinetic analysis to determine the substrate specificities and optimal activity conditions (Aim 1.2). To gain further insight into zymogen (inactive) protease regulation, Aim 1.3 will focus on the mechanism of protease activation. In addition to these studies, we propose to generate a global proteolytic signature of blood fed midguts from the Ae. aegypti mosquito (Aim 2). In parallel with proteomics, this approach will help identify other proteases that have not been previously identified. Taken together, the results of these studies will help lay the foundation for future work in targeting Ae. aegypti midgut proteases and may lead to the development of a new vector control strategy.